This proposal describes a training program for the development of my academic career in cancer biology and professional career as a physician scientist. Throughout the period of this award, I will develop my independent research program. I will also use this time to build on my scientific knowledge and mentoring skills. Dr. Harold Varmus, a leader in the field of cancer biology who has trained numerous successful scientists, will be my mentor. Memorial Sloan-Kettering Cancer Center (MSKCC) will provide institutional support, including ample resources, career development activities and opportunities for interactions between scientists and clinicians to help me achieve my goals. This proposal focuses on lung cancer, the cancer with the highest mortality. Mutations in the epidermal growth factor receptor (EGFR) gene have been associated with sensitivity of lung cancer patients to tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa(R)) or erlotinib (Tarceva). My postdoctoral research in the laboratory of Dr. Varmus over the last three years has led to the identification of numerous targets of mutant EGFR in lung cancer. In this proposal, I propose to extend this work further to understand the signaling pathways that relay the signals from mutant EGFRs to promote cell survival and proliferation and the pathways that are inhibited by TKIs to promote sensitivity. Specific Aim 1:I propose to study the phosphorylation status of proteins downstream of mutated EGFR in human lung cancer cell lines and in variants of these cell lines, which have developed resistance to TKIs upon culture and manipulation in the laboratory. We also propose to study signaling in transgenic mice generated in our laboratory that conditionally express the mutant EGFRs in lung epithelium and generate lung tumors similar to the ones in humans. We will use techniques to enrich phosphorylated proteins, use sensitive mass spectrometry to detect these proteins, and quantify the degree of phosphorylation in these proteins under a variety of conditions, including ligand stimulation and TKI inhibition. Specific Aim 2:I propose to confirm the presence of phosphorylation of targets of mutant EGFR identified in Specific aim 1 by performing immunohistochemistry in lung cancer tissue microarrays (TMAs). I will also use newer specialized mass spectrometry techniques (accurate inclusion mass screening, AIMS and multiple reaction monitoring, MRM) to specifically confirm site-specific phosphorylation of the targets in human lung tumor tissue. Specific Aim 3: I propose to perform functional studies, including experiments to knock-down protein expression by RNAi technology in order to assay survival and proliferation of cells in vitro and in vivo. We also propose to study the cooperation of mutant EGFR and loss of expression of Mig-6, a protein identified to be hyper-phosphorylated on tyrosines in cells expressing mutant EGFR. RELEVANCE: Lung cancer is the leading cause of death from cancer. EGFR mutations account for around 10% of all patients of lung cancer in the US and are associated with sensitivity to tyrosine kinase inhibitors. This proposal is aimed at elucidating the signaling pathways that relay the signals from mutant EGFRs to promote cancer cell survival and proliferation and validate those targets of mutant EGFR in lung cancer patients.